Phospho-eNOS Ser-114 in human mesenchymal stem cells: constitutive phosphorylation, nuclear localization and upregulation during mitosis.

Activity of endothelial nitric oxide synthase (eNOS) is modulated by protein-protein interaction and phosphorylation at specific serine or threonine residues. Using immunofluorescence analysis we show here that proliferating mesenchymal stem cells (MSCs) derived from human bone marrow exhibit cytosolic and pronounced nuclear localization of eNOS. Examination of phosphorylated eNOS subspecies revealed that eNOS phosphorylated at Ser-114 is heavily enriched in the nucleus, whereas eNOS phosphorylated at Ser-1177 is localized at filamentous structures in the cytosol that are abundant in the perinuclear region. Phosphorylation of eNOS at Ser-114 but not at Ser-1177 was strongly increased in cells shortly before mitosis and decreased to normal level after completed cell division. Double immunofluorescence analysis revealed that subcellular localization of 8-hydroxyguanosine immunoreactivity was overlapping with eNOS phosphorylated at Ser-114 in human MSCs providing evidence that phosphorylation at this residue is linked to the generation of superoxide anions. As expected there was only a weak colocalization between eNOS phosphorylated at Ser-1177 and caveolin-1. Different from many other cell systems, human MSCs accumulate eNOS in the nucleus without an acute stimulus. eNOS constitutively phosphorylated at distinct amino acid residues is targeted to different subcellular compartments pointing to an important role of specific phosphorylation events in the life cycle of proliferating human MSCs.

Complications after cryosurgery with new miniature cryoprobes in long hollow bones: an animal trial.

BACKGROUND: In vitro studies show that new miniature cryoprobes are suitable for cryoablation of bone tissue. The aim of this animal trial on 24 sheep was to examine the perioperative complications, particularly the danger of embolism, of cryoablation when using miniature cryoprobes. METHODS: Cryoablations with 2 freeze-thaw cycles each were carried out in the epiphysis of the right tibia and the metaphysis of the left femur. Pulmonary artery pressure (PAP) and central venous pressure (CVP) were measured. Throughout the intra- and perioperative phase, heart rate and oxygen saturation by pulse oxymetry, blood gas and electrolytes were monitored regularly. Postoperative complications were examined up to 24 weeks postoperative. RESULTS: As result, no significant increase of PAP, CVP or heart rate were observed. Blood gases were unremarkable, with pO2 and pCO2 remaining constant throughout the operation. Regarding pH, standard bicarbonate and base excess, only a non-significant shift towards a slight acidosis was seen. There was a mean hemoglobin decrease of 0.5 g/dl. One animal showed postoperative wound infection and wound edge necrosis. No major peri- and postoperative complications associated with cryosurgery of bone were observed, especially regarding clinically relevant pulmonary embolism. CONCLUSION: Surgery with new types of miniature cryoprobes appears to be a safe alternative to or a complement to conventional resection of abnormal bone tissue.

The use of a new miniature cryoprobe for ablation of bone tissue: in vivo assessment of the probe and application of the method to bone in a sheep model.

BACKGROUND: So far, modern miniature cryoprobes were used for local destruction of soft tissue tumours without damaging the adjacent healthy tissue. In this study, cryoablation methodology was applied to bone and the cooling capacity of the probe was examined in vitro and in vivo. METHOD: Freezing was performed by cooling one or two probes (diameter 3.2 mm) to -180 degrees C with liquid nitrogen. The cooling capacity of the probes was determined optically and thermally against a homogeneous reference gel, followed by in vivo measurements on femoral and tibial sheep bone followed by histological examination. RESULTS: Thanks to the synergistic effect, the simultaneous use of 2 probes produced an almost spherical expansion of cold in the homogenous gelatin. During the in vivo freezes, the temperature curves showed a more moderate trend. Nevertheless, due to the synergistic effect, temperatures below -50 degrees C could be reached at a distance of 1 cm from the probe. No local or systemic intraoperative complications were observed. Histological examination revealed cell necrosis up into the -10 degrees C isotherm. CONCLUSIONS: Adequate tissue cooling of the bone matrix can be achieved with in vivo freezes by means of one or more miniature cryoprobes. Therefore, this probe could provide an alternative to or supplement surgical resection of pathological bone processes.